The creation of novel macrolide polyketides has been achieved through genetic manipulation of polyketide synthases. The modular nature of the Type 1 polyketide synthases allows for domain exchange between different polyketide synthase genes, resulting in hybrid genes which produce polyketide synthases with altered properties that result, in turn, in modified macrolide structures. Thus, it is possible to control chain length, choice of chain extender unit, degree of β-carbon oxidation level, and to some degree stereochemistry. The choice of starter unit has been more difficult to control. Two complementary approaches have been described.
Dutton, et al., J. Antibiotics (1991) 44:357–365 demonstrated that the avermectin polyketide synthase was somewhat flexible in choice of starter units. When denied the natural starter unit through inactivation of the branched-chain amino acid dehydrogenase, the avermectin polyketide synthase will accept a variety of α-branched carboxylic acids as the starter unit. However, only about 30 acids out of nearly 800 candidate acids tried were accepted. Acids without an α-branch appear to be metabolized through β-oxidation until an α-branch is reached, further limiting this methodology. Marsden, et al., Science (1998) 79:199–202 exchanged the native loading domain of the erythromycin PKS with that from the avermectin polyketide synthase, resulting in a hybrid PKS having the same loosened starter unit specificity as the avermectin PKS. Clearly, the native specificities of enzymatic domains will always be a limitation on the flexibility of resulting hybrid systems.
A more general method for controlling starter unit specificity has been described by Jacobsen, et al., Science (1997) 277:367–369. Inactivation of the ketosynthase in module 1 (KS1) of the erythromycin PKS (DEBS) results in an enzyme (KS1°-DEBS) incapable of initiating polyketide synthesis using precursors normally available to the cell. When supplied with a suitable thioester of the diketide product of module 1 or its analogs, however, KS1°-DEBS efficiently incorporates these into full-length polyketides. Subsequent experiments have demonstrated that a very wide range of diketide analogs are accepted by KS1°-DEBS, making this a very general method for production of analogs of the polyketide precursor of erythromycin, 6-deoxyerythronolide B (6-dEB), with variations at the positions controlled by the starter unit. Further, this method allows for production of 6-dEB analogs altered at the 12-position; this is equivalent to altering the substrate specificity of the module 1 acyltransferase (AT1) which transfers the first extender unit. While this has been accomplished through the above described domain exchange experiments as well, the “diketide method” allows for introduction of 12-position substituents which are not available from nature. Furthermore, triketide analogs are accepted, opening the 10- and 11-positions of 6-dEB for modification. The 6-dEB analogs obtained can be further converted into analogs of erythromycin by feeding to a suitable converter strain, such as a strain of Saccharopolyspora erythraea containing a non-functional erythromycin PKS. The resulting erythromycins have altered side-chains at the 13-position as well as other optional modifications, and show altered biological activity. These erythromycin analogs can also be produced by introducing the KS1°-mutation into an erythromycin-producing strain of Saccharopolyspora erythraea, then supplying the mutant strain with suitable diketide or triketide thioesters as described above.
Implementation of this method requires the availability of the N-acylcysteamine oligoketide thioesters. Synthetic methods available in the art for these thioesters do not lend themselves to efficient, economical synthesis, or to the systematic production of variants. The diketide and triketides also typically contain chiral centers requiring the control of absolute or relative stereochemistry.
Cane, D. E., et al., J Am Chem Soc (1987) 109:1255–1257 describes a three-step process to produce the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxy pentanoic acid. The method relies on the use of a chiral reagent N-propionyl-(4S)-4-isopropyl-2-oxazolidinone for control of the absolute stereochemistry of the product:
This material results from the acylation of (4S)-4-isopropyl-2-oxazolidinone with propionyl chloride, typically using a strong base such as n-butyllithium at low temperature. The Cane process is an aldol condensation of this starting material with propionaldehyde in the presence of dibutylboron triflate (Bu2BOTf), followed by hydrolysis of the imide (lithium hydroperoxide) and thioesterification with N-acetylcysteamine in the presence of diphenyl phosphorylazide and triethylamine. This multi-step process is inefficient, with substantial losses accompanying the hydrolysis step.
Cane, D. E., et al., J Antibiotics (1995) 48:647–651 was able to improve yields using a five-step process which replaces the aldol condensation with a Claisen condensation between the lithium enolate of the propionyl oxazolidinone (N-propionyl-(4S)-4-benzyl-2-oxazolidinone was used as the stereochemistry controlling starting material in this method) and propionyl chloride followed by reduction of the resulting β-ketoester product using zinc borohydride. Protection of the β-hydroxy substituent as a tert-butyldimethylsilyl ether preceded hydrolysis of the imide, which again required lithium hydroperoxide. The protecting group gave improved yields from hydrolysis, but required an additional two steps to add and remove. This longer process also suffers from the use of zinc borohydride, which is not commercially available.
Cleavage of the N-acyloxazolidinones resulting from either aldol or Claisen condensations as described above is problematic. Various methods of cleavage are known in the art, including that of Evans, D. A., et al., Tetrahedron Lett (1987) 28:6141, in which undesired reaction at the oxazolidinone carbonyl during hydrolysis is suppressed by the use of lithium hydroperoxide. This process requires the use of concentrated solutions of hydrogen peroxide, which are explosive and dangerous for large-scale processes. The N-acyloxazolidinones are unreactive towards thiols or thiolates, although some conversion to thioesters can be observed using concentrated solutions of lithium thiolates in tetrahydrofuran. The low solubility of the thiolates in tetrahydrofuran combined with epimerization of chiral diketides due to the basicity of the thiolates limits the utility of this method. Miyata, O., et al., Syn Lett (1994) 637–638, describes conversion of N-acyloxazolidinones to S-benzylthioesters through the use of lithium benzylthiotrimethylaluminate. The production of more complex thioesters containing groups capable of binding Lewis acids like trimethylaluminum, such as those based on N-acylcysteamine, has not been reported.
The N-acetylcysteamine thioesters of larger oligoketides have also been prepared. Cane, D. E., et al., J Am Chem Soc (1993) 115:522–526 synthesized the N-acetylcysteamine thioester of (4S,5R)-5-hydroxy-4-methyl-2-heptenoic acid using the stereochemically controlled aldol condensation product of N-propionyl-(4S)-4-benzyl-2-oxazolidinone as the starting material:
This imide was converted to the corresponding aldehyde, and extended at the carbonyl group by a Wittig reaction to obtain the desired triketide as the ethyl ester which was then hydrolyzed and converted to the acylcysteamine thioester in a two-step process. Yields were improved by addition of steps protecting the alcohol, Cane, D. E., et al, J Am Chem Soc (1993) 115:527–535. However, this approach clearly does not lend itself to efficient modular solid-phase synthesis since the building of the triketide chain is nonlinear—i.e., the condensations and the Wittig reactions extend the diketide in opposing directions. Each new analog requires complete passage through the synthesis with no common intermediates.
While it is clear that thioester forms of acyl moieties, diketides and triketides can be incorporated by PKS systems, to date, little has been reported concerning the optimal thioesters to produce the desired polyketides other than that N-acetylcysteamine thioesters are generally effective as compared with the free carboxylic acids or their oxy-esters (Cane & Yan). It may be expected, however, that the nature of the thioester, e.g. the acyl group in an N-acylcysteamine thioester, might influence such important factors as water solubility, transport into the bacterial cell, metabolism, and recognition by the PKS. A synthetic method for producing variation in the thioester group itself would thus be advantageous.
The present invention offers both improved efficiency in the synthesis of optically-pure diketide thioester intermediates and an approach which provides for efficient extension of the diketides into the corresponding triketide thioesters and provides for additional condensation steps to extend the oligoketide still further. The present invention further provides a method for synthesis of racemic rather than optically pure diketide thioesters. The racemic materials constitute low-cost alternatives for large-scale production of novel polyketides by fermentation.